There has been much research effort directed to finding natural and synthetic materials having the requisite properties for use as tissue grafts. Surprisingly, it has been found that basement membranes (stroma) prepared from liver tissue of warm-blooded vertebrates by removing cellular components of the liver tissue exhibit certain mechanical and biotropic properties similar to that which has been reported for intestinal submucosal tissue in U.S. Pats. Nos. 4,902,508; 5,281,422; and 5,275,826. Liver basement membranes can be substituted for intestinal submucosa tissue in most, if not all, of the applications previously reported for intestinal submucosa, including enhancing wound healing, promoting endogenous tissue growth, and stimulating cell proliferation and inducing cell differentiation in vivo and in vitro. See U.S. Pat. No. 6,379,710.
The basement membrane of the liver is an extracellular matrix distinct from submucosal extracellular matrices. Liver basement membrane does not support an overlaying mucosa and is devoid of the laminate tissue structure in which submucosal extracellular matrices reside. Liver tissues play a central role in numerous regulatory processes in the body, including glucose metabolism, insulin regulation, anabolic processes for the musculo-skeletal system and central nervous system, and the maintenance of appropriate levels of circulating proteins essential for day to day homeostasis.
U.S. Pat. No. 6,379,710 describes methods for isolating liver basement membranes. However, there has been an ongoing research effort to develop a liver basement membrane preparation/purification protocol that can be used to produce a liver basement membrane preparation in a more pure form (e.g., devoid of cells, cell components, endotoxin, and DNA of liver tissue). The present liver basement membrane preparation can be used to efficiently produce tissue graft materials with more consistent physical properties and more acceptable clinical characteristics.
Accordingly, one embodiment of this invention provides a method for preparing a substantially pure tissue graft composition comprising basement membrane of warm-blooded vertebrate liver tissue. The method comprises the steps of partially hydrolyzing liver tissue by contacting the tissue with an aqueous composition comprising an exogenous protease, washing the liver tissue with an aqueous detergent composition comprising a non-denaturing detergent, removing the non-denaturing detergent, and washing the liver tissue with a composition comprising a denaturing detergent substantially free of non-denaturing detergent. The graft composition so prepared comprises an extracellular matrix consisting essentially of liver basement membrane devoid of endogenous cells associated with the liver tissue. In another embodiment, a liver basement membrane composition prepared according to this method is provided.
The graft composition so prepared is useful for inducing the formation of endogenous tissue at a site in need of endogenous tissue growth in a warm-blooded vertebrate. Thus, another embodiment of this invention is a method comprising the step of implanting into the vertebrate the graft composition prepared as described above comprising basement membrane of liver tissue of a warm-blooded vertebrate in an amount effective to induce endogenous tissue growth at the site of administration of the graft composition.
The liver basement membrane graft composition prepared in accordance with this invention can be used to manufacture a non-immunogenic tissue graft composition for use in the repair of damaged or diseased tissues. The tissue graft composition comprises the basement membrane of liver tissue of a warm-blooded vertebrate substantially free, preferably devoid, of endogenous cells (e.g., hepatocytes and bile duct cells) and cell components of the warm-blooded vertebrate and devoid of endotoxin and DNA. The tissue graft composition can be implanted, or fluidized and injected, into a vertebrate host to contact damaged, defective, or missing tissues and induce the repair or replacement of the tissues. The tissue graft composition can also be applied as a component of a wound dressing (ointment or bandage) in fluidized or solid form for topical application to promote wound healing.
The liver tissue derived graft composition of this invention can also be utilized as a cell growth substrate for growing cells in vitro. Thus, in another embodiment, a liver tissue derived basement membrane composition for supporting the growth of a cell population is provided. The composition comprises the liver basement membrane composition described above, wherein the liver basement membrane is devoid of source liver tissue endogenous cells, and added nutrients to support the growth of the cell population in vitro. In an alternate embodiment, a liver tissue derived basement membrane composition for supporting the growth of a cell population is provided. The composition comprises culture-ware coated with a matrix comprising the liver basement membrane composition prepared as described above wherein the liver basement membrane is devoid of source liver tissue endogenous cells.